Hedgehog (Hh) growth factor signaling is one of the principal pathways regulating tissue patterning in embryogenesis, best known in vertebrates for its essential roles in nervous system and limb development. Even modest perturbations in Hh signaling have been linked to developmental and cognitive defects in humans. Moreover, Hh also functions in regulating growth and differentiation of adult stem cells, so that defects in Hh pathway signaling have been causally linked to many types of cancers, including Basal Cell Carcinoma, and cancers of the pancreas, intestine, and nervous system. Understanding the requirements for and regulation of Hh signaling has important significance for the maintenance of human health and development of therapies. We have recently discovered an unexpected and previously undescribed requirement for the cellular response to Hh signaling. In the absence of normal function of the Ryanodine Receptor intracellular Calcium Release Channel (CRC), several cell types whose development is known to depend directly on Hh signaling in the zebrafish embryo fail to arise. In embryos deficient in CRC function, tissue precursor cells that normally sense and respond to Hh signals fail to upregulate genes, including patched, that are the earliest known markers of the Hh response pathway. As preliminary findings indicate that it is the responding cells that require CRC activity and that these cells do not die in the absence of CRC activity, we hypothesize that regulated calcium mobilization is required for cells to sense or to execute the response to the Hh growth factor. Because these findings are novel and are not anticipated by any prevailing model of Hh pathway function, and because the regulation of Hh pathway signaling is so important for human health, we seek to use the R21 Exploratory/Developmental Research Grant Program ("high risk - high impact") mechanism to further define the role of intracellular calcium mobilization in Hedgehog signaling. First, we will determine whether the requirement for CRC function is a general feature of the response to Hh signaling in the zebrafish embryo by measuring the effects of blocked CRC activity on the development of multiple Hh-dependent tissues. Second, we will determine whether Hh-signaling and/or Hh-receiving cells require CRC function. Third, we will begin to identify the specific role of calcium mobilization within the Hh signal transduction pathway. Results from these experiments will establish whether CRC activity is needed to transmit, receive, or respond to Hh signaling, and thus will form the basis of our future research directions. PUBLIC HEALTH RELEVANCE: Hedgehog (Hh) growth factor signaling is one of the principal intercellular signaling pathways controlling cell growth and differentiation. Defects in the regulation of Hh signaling contribute to both cancers and birth defects in humans. Understanding the requirements for and regulation of Hh signaling has important significance for the maintenance of human health and the development of disease therapies. This proposal investigates how the controlled release and mobilization of calcium within the cell contributes to the regulation of Hh signaling.